The Photodynamic Therapy (PDT) is a therapeutic modality of clinical use widely used for the treatment of various skin diseases, including cancer.
PDT is based on the exogenous administration of photosensitive compounds (PS) or precursors of the same which accumulate by means of different mechanisms in a preferred manner in the target tissues. The irradiation of the tissue with light of appropriate wavelength, usually in the red region of the spectrum (λ≥600 nm) for greater penetration into the tissue, and in the presence of intracellular oxygen induces the production of Reactive Oxygen Species (ROS), especially singlet oxygen. The rapid accumulation of intracellular ROS above a critical threshold promotes a strong photosensitization inducing cell death.
The 5-aminolevulinic acid (ALA) and, to a greater extent, its methylated derivative methyl aminolevulinate (MAL), are two of the most used compounds in a clinical setting, in dermatological protocols with PDT. Their low molecular weight determines a high absorption through the epidermis allowing topical application of the same. These compounds are not photoactive by themselves, but act as precursors of the endogenous PS protoporphyrin IX (PpIX). Once absorbed by the cell, they are incorporated in the metabolic pathway of the biosynthesis of heme group promoting an abnormal accumulation of PpIX that can last between hours and days, with the subsequent photosensitization of the target tissue. The MAL-PDT treatment is widespread in clinical dermatology, particularly for the treatment of actinic keratosis and basal cell carcinoma.
It has been described that the experimental treatment with exogenous sources of ROS in low amounts, such as hydrogen peroxide, can promote cell proliferation in in vitro cultures (Boonstra J, Post J A. “Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells”. Gene 337: 1-13, 2004), including potential neural progenitor cells grown by the system of neurospheres (Le Belle J E et al. “Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt- dependant manner”, Cell Stem Cell. 8: 59-71, 2011).
However, there is so far no experimental evidence to suggest a causal relationship between endogenous production of ROS in a tissue and the functional activation of a type of stem cell contained in said tissue involving physiological consequences, with potential clinical, pharmacologic or cosmetic use. This is largely because there is no in vivo experimental procedure that allows inducing a controlled endogenous production of ROS in a tissue.
There are also no experimental data indicating that endogenous ROS accumulation in tissues can be part of a normal homeostatic process functionally dependent on stem cells. On the contrary, all the experimental results showing an in vivo accumulation of ROS in tissues indicate that this accumulation is abnormal and is associated with pathologic conditions and aging processes (Valko M et al. “Free radicals and antioxidants in normal physiological functions and human disease”, Int. J. Biochem. Cell Biol. 39: 44-84, 2007). Also, the topical treatment of a tissue with exogenous sources of ROS can not be considered in any way biologically equivalent to a physiological production of endogenous ROS.
The inventors describe an experimental method that uses PDT-MAL to induce endogenous production of ROS in the hair follicle capable of activating the epidermal stem cells contained in this niche. This stimulation of the epidermal stem cells is due to the transcriptional activation by ROS in the target tissue of the genes of the prolactin family 2, also known as proliferins, particularly proliferin-2 or Pr12c3. Taking into account that there has been proposed a potential role for Pr12c3 in the in vitro expansion of hematopoietic stem cells (Choong M L et al. “A novel role for proliferin-2 in the ex vivo expansion of hematopoietic stem cells”, FEBS Lett. 550: 155-62, 2003), the in vivo stimulation of genes of the proliferin family by ROS associated with an activation of epidermal stem cells is a surprising and important discovery in its own right.
In the current art, one of the most widespread uses of the epidermal stem cells in the field of bioengineering is the generation of skin equivalents of epidermal or dermo-epidermal component (Shevchenko R V et al. “A review of tissue-engineered skin bioconstructs available for skin reconstruction”, J. R. Soc. Interface 7: 229-58, 2010). These skin equivalents or artificial skins have very important applications in regenerative medicine, primarily for the treatment of burns and wounds of great extent and depth. The ideal treatment for this type of injury is the autograft with different types of skin equivalents generated from skin of the own patient. Given the restrictions in the European Union (Directive 2010/63/EU) and other countries for the use of experimental animals, another key application of skin equivalents is their use as biological models to test the feasibility and toxicity of pharmaceutical and cosmetic compounds.
In this type of applications an essential limitation is the generation time of a functional skin equivalent, which requires the ex vivo expansion of the epidermal progenitors and the stratification of the epidermal component in contact with air. In a clinical setting, the excessively long time in the production of equivalents for autograft pose an immediate danger to the patient, which requires the use of alternative therapies such as cadaver skin grafts or little humanized synthetic equivalents. In pharmacology and cosmetics, the generation time of artificial skin is directly related to its production cost. Therefore, a problem that arises in the art is to develop an experimental method to speed up these processes during the formation of the skin equivalent.
On the other side, different experimental evidence indicates that many skin diseases, including different types of cancer, alopecia and processes such as aging may be due to a defect in the activity of epidermal stem cells, and in particular of those residing in the niche of the hair follicle. A major limitation in the clinical management of these diseases is that the treatments currently available are symptomatic and nonspecific, that is, they are not intended to functionally modulate the activity of the epidermal stem cells.
The problem that arises in the art, therefore, is to develop methods more specific and effective than the current methods to generate the skin equivalents and treat diseases related to stem cells. The solution provided by the present invention is a treatment by means of a PDT method capable of inducing the endogenous production of ROS and the activation of epidermal stem cells.